The present invention relates to a process for purification of the active principle of the fruit of the plant Synsepalum dulcificum. Also disclosed is a method for the use of this active principle in the control of obesity.
The botanical plant species of Synsepalum dulcificum is indigenous to tropical West Africa, where it is often referred to as "miracle fruit." The plant, which grows in the form of a shrub, yields ripe red berries from December to June, the berries being ellipsoidal in shape and about 0.75 inch long, and composed of a thin layer of pulp surrounding a single large seed. These berries have the unique property of modifying the taste of sour foods to make such foods taste sweet after the fruit pulp has been chewed.
Previous methods for the purification of the active principle of plants such as Synsepalum dulcificum have been characterized by a complex series of steps involving bulky equipment and long periods of time for the separation of an active principle which in many cases has been of a low degree of purity, often containing contaminants such as proteases and tannins in relatively large amounts. Such prior art methods include those described by Inglett et al., J. Agri. Food Chem., 13:284 (1965), Brouwer et al., Nature 220:373 (1968) and Beidler et al., Science 161:1241 (1968). The method of Inglett et al. involves purification of the active principle of miracle fruit in phases A and B, with phase A involving successive treatment of the plant parts with petroleum ether, chloroform, ethyl acetate, acetone, absolute ethanol and water, and phase B including successive treatment with water, aqueous alcohol, absolute alcohol, acetone, chloroform and n-hexane, followed by hydrolysis of fractions A and B. By removal of inactive matter, a five-fold concentration of the active material was achieved.
Brouwer et al. purified the active principle by ammonium sulfate fractionation and gel chromatography of the crude extract while Beidler et al. passed the crude extract through a column of diethylaminoethyl-Sephadex followed by ion-exchange chromatography on a carboxymethyl-Sephadex column.
By the present invention, there is provided a process for the purification of the active principle of the fruit of the plant Synsepalum dulcificum which is simple in nature and capable of being carried out in a short period of time to provide a product having a high degree of purity. The present method is accomplished with fewer steps than prior art processes and proteases and tannins are substantially completely eliminated. The presence of such contaminants in prior purification methods has resulted in destruction of the active principle by a process of enzymatic degradation and is a chief reason for the approximately two-fold increase in the activity of the principle prepared by the method of the present invention as compared with prior methods.
In general, an extract containing the desired active principle is obtained by extraction in a basic medium with polyvinyl pyrrolidone. The concentrated extract is acidified after addition of .epsilon.-amino caproic acid with glacial acetic acid. The filtered solution containing the active principle is chromatographed on a carboxymethyl polyacrylamide gel adsorbent, employing liquid column chromatography techniques, following which the active principle is eluted with sodium phosphate. The active principle is then placed on a column of QAE-Sephadex A-50, an ion exchange medium of diethyl-(2-hydroxypropyl) aminoethyl groups coupled to a modified dextran matrix. The purpose of this step is to purify further the active principle by placing it through a cellulose or polyacrylamide-based strongly basic anion exchanger. The active principle is eluted from the column and may then be subjected to a third liquid column chromatography procedure on a column of carboxymethyl polyacrylamide gel. The active principle is again eluted with sodium phosphate to provide the final purified product.
It has been found that this active principle, prepared in accordance with the present invention, is very useful in controlling obesity when administered orally. Various pharmacological studies have been performed using the active principle of Synsepalum dulcificum as obtained by the present invention and through these studies the preferred and safe dosage levels have been determined. The active principle, often called "MFP" (miracle fruit principle) or "miraculin", is effective in controlling obesity at concentrations of 10.sup.-.sup.7 M.